kinesin: how it waits between steps harvard biovisions: “the inner life of the cell” holly...

Kinesin: How it Waits Between Steps

Harvard Biovisions: “The Inner Life of the Cell” http://multimedia.mcb.harvard.edu/

Holly Durst

What is Kinesin?What is Kinesin?• dimeric motor protein

• carries cellular cargo along microtubules by hydrolyzing ATP

• takes several hundred “steps” along a microtubule without detaching

Harvard Biovisions: “The Inner Life of the Cell” http://multimedia.mcb.harvard.edu/

1. ATP binding to leading head initiates neck linker docking and the other head is thrown forward

2. New leading head docks onto binding site after diffusional search, resulting in 80 Å movement of attached cargo

3. This accelerates ADP release and trailing head hydrolyzes ATP to ADP-Pi

4. ATP binds to leading head

α β

Coiled coil

Neck linkers

FRETFRET

http://bio.physics.uiuc.edu/images/FRET_concept.jpg

Objective

• Use a series of smFRET experiments to detect whether kinesin is bound to its microtubule track by one or two heads in its ‘waiting’ conformation between steps

How kinesin waits between stepsHow kinesin waits between steps

Teppei Mori, Ronald D. Vale & Michio Tomishige

• ‘cysteine light’ human ubiquitous kinesin-1 dimer which cysteine residues and/or mutations were introduced into

• Dye-labelled kinesins were imaged moving along sea-urchin axonemes with a custom-built prism-type laser-illuminated total-internal reflection fluorescence microscope

The PlayersThe Players

The PlayersThe Players

1. Heterodimer • one chain containing

single cysteine residue in plus end oriented tip of core (residue 215)

• one chain containing single cysteine residue in minus-end oriented base of the core (residue 43)

Used two FRET Sensors:

2. Homodimer with a cysteine residue in both chains at the beginning of the neck linker (residue 324)

The PlayersThe Players

• Donor dye: Maleimide modified Cy3

• Acceptor dye: Maleimide modified Cy5

• Molecules that contained both dyes were selected for smFRET observations

Testing the SensorsTesting the Sensors

Testing the SensorsTesting the Sensors• Examined FRET efficiency in presence of non-

hydrolyzable nucleotide analog AMP-PNP so that both kinesin heads are bound statically to the microtubule

Testing the SensorsTesting the Sensors

• Bimodal distribution of low (10%) and high (90%)

• As expected if two kinesin heads are bound to adjacent tubulin subunits 8 nm apart

• High peak – 43 dye on leading head and 215 dye on trailing head

• Low peak – 215 dye on leading head and 43 dye on trailing head

• Unimodal distribution centered at about 35%

• Is consistent with a two-head bound state

smFRET Efficiencies for 215-43

smFRET Efficiencies for 324-324

Binding along single protofilament supported by experiments with a 149-324 sensor

Different ConditionsDifferent Conditions

• Low ADP concentrationsRemember: ADP occupying binding site

= weak microtubule binding

Different ConditionsDifferent Conditions

215-43 Sensor unimodal at about 30%

324-324 Sensor shift from 35% to 60%

• kinesin heads come closer together

Under these conditions, these distributions reflect a one-head bound state

• Low ADP plus excess inorganic phosphate

Different ConditionsDifferent Conditions

• Partial occupancy of an ADP·Pi state in tethered head

215-43 Sensor

324-324 Sensor

Peaks characteristic of a two-head bound state• Similar results for addition of AlF4-

• Both heads nucleotide free

Different ConditionsDifferent Conditions

215-43 Sensor

324-324 Sensor

• distributions similar to AMP-PNP, but with broader distributions

Nucleotide-free kinesin primarily adopts a two-head bound state with partial occupancy of a one-head-bound state

FRET efficiency trace of individual axoneme-bound 215-43 heterodimer kinesin

• Signal of 215-43 in AMP-PNP was fairly constant

• A subset of molecules with ADP or ADP/Pi or under nucleotide-free conditions underwent abrupt FRET transitions

• Unbinding and rebinding of kinesin head with microtubule

• Mutated so only one head could bind to microtubules under all conditions– Y274A/R278A/K281A in loop 12 (L12-triple)

1. 215(WT) – 43 (L12)

2. 215(L12) – 43 (WT)

MutationsMutations

MutationsMutations

• 200 nM ADP • 215(WT)-43(L12) and 215(L12)-43(WT) both

produced unimodal distributions centered at about 30%

• Distances between 43-labelled and 215 labelled dyes are similar

• Similar result for nucleotide-free state

MutationsMutations

• Addition of AMP-PNP• 215(WT)-43(L12) – bimodal with primary peak at

80%• 215(L12)-43(WT) – major peak shifted in opposite

direction toward lower efficiencies• movement of L12 triple towards plus-end oriented

tip of bound head

MutationsMutations

• 215/342 dyes on wild-type chain to probe neck linker conformations in the bound head

MutationsMutations

MutationsMutations

Translation of unbound head from rear position to forward position is driven by nucleotide-dependent docking of neck-linker

• Saturating ATP concentration (1 mM)– Can only measure an average

Dynamic MeasurementsDynamic Measurements

• 215-43 showed broad distribution centered at about 50%

• Average of bimodal 10%, 90% FRET distribution of static two-head bound kinesin

• Different from 30% value of one-head bound kinesin

Dynamic MeasurementsDynamic Measurements

• 324-324 unimodal distribution centered at about 30%

Kinesin spends most of the time bound with two heads to the microtubule when moving at saturating ATP concentrations

Dynamic MeasurementsDynamic Measurements

• Subsaturating ATP concentration (2 μM)

Dynamic MeasurementsDynamic Measurements

• 215-43 shifted to about 30%

• 324-324 shifted to about 60%• More similar to 200 nM ADP (one-head bound)

Suggests that kinesin waits primarily as a one-head bound intermediate when ATP binding becomes the rate-limiting step in the ATPase cycle

Dynamic MeasurementsDynamic Measurements

• Longer dwell times at low ATP concentration

Dynamic MeasurementsDynamic Measurements

• Spent most time in a roughly 30% FRET state (one-head bound) with brief spikes towards higher (80%) FRET values

• Higher values represent transient two-head bound intermediate state

• Transitions from 30% to lower FRET state should also occur

• Difficult to distinguish from noise

Dynamic MeasurementsDynamic Measurements

• Dwell-time histogram best fitted by a convolution of two exponentials

• Two rate-limiting ATP binding events occur between the two high-FRET spikes

Dynamic MeasurementsDynamic Measurements

• Mean dwell time (140 ms) is comparable to predicted dwell time

• Total number of spikes divided by displacement of these molecules yielded an average distance of about 17 nm per spike

• Close to double kinesin step size

Dynamic MeasurementsDynamic Measurements

• A kinesin step at low ATP concentrations involves a short-lived, two-head bound state, which then undergoes a transition to a longer-lived, one-head bound state

Dynamic MeasurementsDynamic Measurements

• At high ATP concentration (the rate-limiting step is the detachment of the trailing head triggered by ATP hydrolysis/phosphate release), kinesin moves quickly from one two-head bound state to the next

• At low ATP concentration (ATP binding to the leading head is rate-limiting), the trailing head releases its Pi and detaches from the microtubule, producing a long-lived one-head bound state

SummarySummary

SummarySummary

• Kinesin waits as either a one-head bound or two-head bound intermediate, depending on ATP concentration and the rate-limiting step

DiscussionDiscussion

• ATPase cycles in the two kinesin heads are coordinated during processive motion

• Gating model proposes that detached head waits in front of bound head and is in a conformation that prevents it from binding tubulin

• But, transient interactions with the microtubule are seen

• Additional mechanism must keep detached head from progressing through ATPase cycle until its partner binds ATP

DiscussionDiscussion

• Detached head will not release ADP when it is interacting with rear tubulin-binding site

• ADP release could occur after the bound head binds ATP and docks the neck-linker, translating the detached head to a forward tubulin-binding site

• Results are supported by Guydosh and Block who showed that nucleotide dissociation occurs only when a head is in the forward position

• Position dependence controlled by conformation of neck-linker

DiscussionDiscussion

• How does the conformation of the neck-linker affect transitions in the ATPase cycle?

Future WorkFuture Work

ReferencesReferences• Mori, T.; Vale, R. D.; Tomishige, M. Nature 2007, 450,

750-754• Vale, R. D.; Milligan, R. A. Science 2000, 288, 88-95

QUESTIONS?